Recombinant Human ME2 Protein: Handling & Application Protocol

Recombinant Human ME2 Protein is a mitochondrial NAD(P)+-dependent malic enzyme used in metabolic flux studies, cancer biology research, and enzyme kinetics assays requiring a highly purified, biologically active human ME2 source.

Product SKU

rp371711

Protein Name

Recombinant Human Malic Enzyme 2 (ME2)

Enzyme Class

Mitochondrial NAD(P)+-dependent malic enzyme

Primary Application

Enzyme activity assays, metabolic research, cancer biology studies

Recommended Storage

-80 °C for long-term storage; avoid repeated freeze-thaw cycles

Reference Source

Absea #PP-5832

Materials Required

Before beginning, ensure the following materials are available:

• Recombinant Human ME2 Protein (Aladdin Scientific, SKU: rp371711)
• Sterile, nuclease-free deionized water or the reconstitution buffer recommended in the product datasheet
• Microcentrifuge tubes (low-binding, 1.5 mL)
• Micropipettes and calibrated tips
• Dry ice or liquid nitrogen for handling
• -80 °C freezer for storage
• Spectrophotometer or plate reader capable of measuring absorbance at 340 nm (for NADH/NADPH-based enzyme activity assay)
• Assay buffer: typically 50 mM Tris-HCl pH 7.4, 5 mM MgCl₂, 0.5 mM NAD⁺ or NADP⁺, 1 mM L-malate

Note: Exact buffer composition and reconstitution conditions should be verified against the official product datasheet provided with the lot.

Reconstitution and Storage

Follow these steps to reconstitute and store Recombinant Human ME2 Protein correctly:

1. Thaw carefully: Allow the vial to thaw on ice at 4 °C. Do not thaw at room temperature or use a water bath, as this may reduce protein activity.
2. Centrifuge: Briefly centrifuge the vial (1,000–2,000 × g, 30 seconds) to collect any lyophilized material from the walls and cap.
3. Reconstitute: Add the volume of sterile deionized water or the recommended reconstitution buffer specified in the datasheet directly to the vial. Gently pipette up and down or swirl — do not vortex — until fully dissolved.
4. Aliquot: Prepare single-use aliquots immediately after reconstitution to minimize freeze-thaw cycles. Use low-binding microcentrifuge tubes.
5. Short-term storage: Store at 4 °C for up to 1 week if the protein will be used within that period.
6. Long-term storage: Store aliquots at -80 °C. Avoid repeated freeze-thaw cycles, as each cycle may significantly reduce enzymatic activity and protein integrity.
7. Carrier protein: If the protein is supplied at a very low concentration, consider adding a carrier protein (e.g., 0.1% BSA) as directed by the datasheet to maintain stability.

Review flag: Specific reconstitution volume, buffer formulation, and stock concentration should be confirmed from the official lot-specific datasheet (Absea #PP-5832).

Enzyme Activity Assay Protocol

ME2 catalyzes the oxidative decarboxylation of L-malate to pyruvate with concomitant reduction of NAD⁺ or NADP⁺ to NADH or NADPH, which can be monitored spectrophotometrically at 340 nm.

Procedure

1. Prepare assay buffer: Use 50 mM Tris-HCl (pH 7.4) containing 5 mM MgCl₂. Warm to 37 °C before use.
2. Prepare substrate solution: Dissolve L-malate to 1 mM and NAD⁺ (or NADP⁺) to 0.5 mM in assay buffer. Adjust pH if necessary.
3. Set up reaction: In a cuvette or microplate well, combine substrate solution (950 µL or appropriate volume) with Recombinant Human ME2 Protein at the concentration recommended in the datasheet. Keep on ice until ready.
4. Initiate reaction: Transfer to 37 °C and immediately begin absorbance recording at 340 nm. Record readings every 30 seconds for at least 5 minutes.
5. Calculate activity: Use the molar extinction coefficient of NADH/NADPH (ε = 6,220 M⁻¹cm⁻¹) to convert ΔA₃₄₀/min to enzyme activity (nmol/min/µg protein).
6. Controls: Include a no-enzyme blank and a no-substrate blank for baseline correction.

Review flag: Optimal ME2 protein concentration, reaction volume, and specific activity values are not provided in the source material and should be verified from the datasheet or relevant published literature.

Tips, Troubleshooting & Safety

Tips for best results:

• Always keep the protein on ice during handling to preserve activity.
• Use freshly prepared substrate solutions; NAD⁺ and NADP⁺ solutions can degrade over time.
• Pre-equilibrate all reagents to assay temperature before combining.
• Use low-binding plasticware to minimize protein adsorption losses.

Troubleshooting:

Problem	Possible Cause	Solution
Low or no signal at 340 nm	Inactive protein due to improper storage or freeze-thaw	Use freshly reconstituted protein; ensure -80 °C storage
High background	Contamination of substrates or buffer	Use fresh reagent-grade chemicals; include proper blanks
Non-linear kinetics	Substrate depletion or inhibitor present	Reduce protein concentration or reaction time; check reagent purity

Safety: Handle Recombinant Human ME2 Protein as a potential biological hazard. Wear appropriate PPE (gloves, lab coat, eye protection). Dispose of protein-containing waste according to your institution's biosafety guidelines. This product is for research use only and is not intended for therapeutic, diagnostic, or in vivo use.

Frequently asked questions

What is the biological function of human ME2 protein?

ME2 (Malic Enzyme 2) is a mitochondrial enzyme that catalyzes the oxidative decarboxylation of L-malate to pyruvate, coupled with the reduction of NAD⁺ or NADP⁺ to NADH or NADPH. It plays a central role in linking the TCA cycle to biosynthetic pathways and energy metabolism, and has been implicated in cancer cell metabolic reprogramming.

How should I avoid loss of ME2 protein activity during experiments?

To preserve activity, always keep the protein on ice during handling, avoid repeated freeze-thaw cycles by using single-use aliquots, do not vortex the protein solution, and use low-binding labware. Reconstitute only the amount needed for immediate use and store unused aliquots at -80 °C.

Can Recombinant Human ME2 Protein be used in Western blot or ELISA applications?

Recombinant Human ME2 Protein is primarily intended for enzyme activity assays and biochemical studies. It may also serve as a positive control antigen in Western blot or ELISA experiments where an ME2 antibody is being validated. Please consult the product datasheet for confirmed validated applications for this specific lot.

What cofactors are required for ME2 enzymatic activity?

ME2 requires a divalent metal ion cofactor, typically Mg²⁺ or Mn²⁺, for catalytic activity. The enzyme can utilize either NAD⁺ or NADP⁺ as the electron acceptor, with a preference for NAD⁺. Ensure these cofactors are present at appropriate concentrations in your assay buffer.

References

1. Absea Biotechnology. Recombinant Human ME2 Protein, Product No. PP-5832. Product datasheet.
2. Chang GG, Tong L. Structure and function of malic enzymes, a new class of oxidative decarboxylases. Biochemistry. 2003;42(44):12721-12733. doi:10.1021/bi035251+
3. Ren JG, Seth P, Clish CB, et al. Knockdown of malic enzyme 2 suppresses lung tumor growth, induces differentiation and impacts PI3K/AKT signaling. Sci Rep. 2014;4:5414. doi:10.1038/srep05414

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.